1. Field of the Invention
The present invention is directed to methods and systems for stabilizing arachidonic acid (AA), and more particularly to methods and systems for stabilizing AA in a single use platelet function assay.
2. Description of the Related Art
The role of platelets in mammalian physiology is extraordinarily diverse, but their primary role is in promoting hemostasis. In many situations, an evaluation of the ability of blood to clot is desired, a parameter that is frequently controlled by the ability of platelets to adhere and/or aggregate. Of interest, therefore, is the assessment of the adhesive functions of platelets. For example, questions of interest include whether to administer drugs that will block, or promote, clot formation, or whether to detect deficiencies in platelet function prior to surgical procedures. Also of interest is evaluating the effectiveness of a platelet inhibitor that is being tested as a new drug or is being used as approved clinical treatment in a patient.
Platelet aggregation plays a key role in the pathogenesis of thrombosis and acute coronary artery disease. Evidence suggests that significant platelet function variability exists in the response to various anti-platelet agents. In particular, aspirin is widely used for its anti-platelet effects in patients with acute coronary syndromes (ACS). The clinical benefits of aspirin in ACS is due in part to its ability to inhibit thromboxane A2 (TXA2), known to cause platelet aggregation, by the irreversible acetylation of the cyclo-oxygenase 1 (COX-1) enzyme.
Platelet aggregation is a term used to describe the binding of platelets to one another. In vitro platelet aggregometry is the laboratory method used to assess the in vivo ability of platelets to form the aggregates leading to a primary hemostatic plug. In this technique an anti-coagulated whole blood sample is centrifuged under multiple conditions to create both a platelet-rich plasma (PRP) and platelet-poor plasma (PPP) sample. An aggregating agent is then added to the PRP and aggregation of platelets monitored optically while in parallel with this, a separate optical measurement is made using the PPP sample. The percent aggregation is then determined by use of the PPP channel as the 100% aggregation reference level to compare with the PRP channel.
Helena Laboratories (Beaumont, Tex.) a manufacturer of platelet aggregometry systems for laboratory use provides educational literature that suggests the appropriate aggregating agent depending upon the purpose of the test. For assessing the effects of aspirin on platelet function, Helena Laboratories states, “The arachidonic acid platelet aggregation assay is the only practical way to monitor the effects of aspirin therapy, now widely used to prevent strokes and heart attacks.” Helena Laboratories, Evaluation of Platelet Function Wall Chart 586–25. Arachidonic acid is a fatty acid present in the granules and membranes of human platelets. Marcus A J: Platelet lipids. In Coleman R W, Hirsh J, Marder V J, Salzman E W: Hemostasis and thrombosis: Basic principles and clinical practice, pg 472. JB Lippencott Company, Philadelphia 1982. It is liberated from phospholipids and, in the presence of the enzyme cyclo-oxygenase one (COX-1), incorporates oxygen to form the endoperoxide prostaglandin G2 (PGG2). PGG2 is then quickly transformed to prostaglandin H2 (PGH2) which in turn is converted to thromboxane A2 a potent inducer of platelet aggregation. Ingestion of aspirin or aspirin-containing compounds inhibits COX-1 mediated oxygen consumption, thus precluding all subsequent events leading to platelet aggregation. Bye A, Lewis Y, O'Grady J: Effect of a single oral dose of aspirin on the platelet aggregation response to arachidonic acid. Br J Clin Pharmac 7:283, 1979.
In vitro addition of arachidonic acid to normal platelet rich plasma results in a burst of oxygen consumption, thromboxane formation and platelet aggregation. Moncada S, Vane J R: Arachidonic acid metabolites and the interactions between platelets and blood vessel walls. N Eng J Med 300:1142, 1979. However, in the presence of aspirin or aspirin-containing compounds, these reactions are absent. Ingerman C M, Smith J B, Shipiro S, Sedar A, Silver A, Silver M J: Hereditary abnormality of platelet aggregation attributable to nucleotide storage pool deficiency, Blood 52:332, 1978.
The challenge with the use of arachidonic acid in clinical settings is the relatively unstable nature of the compound. When exposed to oxygen, arachidonic acid undergoes a process called autoxidation. Autoxidation is generally defined as a chemical reaction that usually takes place at ambient temperature between atmospheric oxygen and an organic compound. Common examples of the autoxidation phenomena are the browning of fruit, rusting of metal, and the degeneration of rubber products. Autoxidation causes arachidonic acid to turn yellow and deteriorate rapidly. In typical laboratory use, arachidonic acid is stored at −20° C. in sealed inert ampoules and once thawed, recommended to be used within 24 hours. Sigma-Aldrich Data Sheets A9673 and A8798. Alternatively, some manufacturers lyophilize a salt-based version of arachidonic acid but again the material must be stored in a sealed inert ampoule at −20° C. and used immediately upon opening. In the clinical setting there is often little advance notice of the need to run a particular test and the necessity to manage the amount of material to thaw and the subsequent use of the material in a timely manner is both cumbersome and time consuming.
Another aspect of the autoxidation of arachidonic acid, of particular relevance to its use as a platelet activator, is that the ex vivo autoxidation of arachidonic acid does not necessarily create the same by-products as in vivo oxidation and in some cases can produce stable by-products that mimic TXA2. Use of arachidonic acid that had degraded in this manner in an assay to assess the effects of aspirin would falsely indicate that aspirin was having no effect on platelet aggregation.
The autoxidation phenomena could be prevented by the total exclusion of oxygen or other oxidizing substances but this is generally not practical. Instead, what is more typically done is to utilize inhibitors that decrease the reaction rate or prolong the induction period. However complete prevention of autoxidation is unlikely. Substances that can suppress autoxidation are termed inhibitors or antioxidants. Preventive inhibitors decrease the rate of autoxidation by suppressing the rate of initiation reactions. Antioxidants in the true sense are substances that can inhibit propagation steps; that is, they interrupt autoxidation chain reactions because after giving up their electron they are still in a stable configuration. Antioxidants are commonly used in food preservation to keep foods from becoming rancid, browning, or developing black spots. Antioxidants also minimize the damage to some essential amino acids and the loss of some vitamins. The two common types of antioxidants used in foods are acids and phenolic compounds. Examples of acid antioxidants are ascorbic and citric acids, while phenolic antioxidant compounds include BHA, BHT, TBHQ, Tocopherols, Lecithin, THBP, Gum and Glycine.
Because of the difficulty with storage and handling, no clinical platelet function analyzer currently utilizes arachidonic acid to measure platelet response to aspirin. Instead, systems like the Dade Behring PFA-100® or Plateletworks® use a combination of platelet activators such as adenosine diphosphate (ADP), collagen and epinephrine. These systems however have shown poor sensitivity and specificity to the detection of aspirin impaired platelet function since their activation agonists are not specific to the pathway targeted by aspirin. The initial Accumetrics' VerifyNow™ Aspirin Assay used cationic propyl gallate (cPG) as the platelet agonist. cPG activates platelets by causing the release of platelet bound arachidonic acid from the phosopholipid layer and has been shown to provide a sensitive and specific activation of platelets along the pathway targeted by aspirin. Steiskal, et al, Application of Cationic Propyl Gallate as Inducer of Thrombocyte Aggregation For Evaluation of Effectiveness of Antiaggregation Therapy. The limitation of cPG however is that when used in whole blood versus PRP the activation of platelets is less consistent presumably due to the red blood cell's effect on the cationic charge.
A rapid platelet function assay has recently been developed and is described in U.S. Pat. No. 5,763,199. The assay determines glycoprotein (GP)IIb/IIIa receptor blockade in undiluted whole blood. Agglutination of small polymeric beads coated with a GPIIb/IIIa ligand such as fibrinogen results when the beads are contacted with whole blood containing platelets with activated GPIIb/IIIa receptors that are not blocked. Failure to agglutinate indicates either failure of the GPIIb/IIIa receptors to become activated and/or blockade of the GPIIb/IIIa receptors. In a preferred embodiment, the addition of a platelet activator like arachidonic acid, results in an assay that is rapid and convenient enough to be performed at the bedside and that results in agglutination of the small polymeric beads within a convenient, known period of time if the activation receptors are not blocked. The assay includes the ability to transfer blood to be tested from a collection container to an assay device without opening the collection container.
There is a need for a method for rapidly determining the level of platelet inhibition in whole blood, due to aspirin usage, with a single use arachidonic based assay device that can be stored at room temperature for many months.